Wet transfer: western blot - (Jul/26/2012 )

Hi..This is my first time doing wet protein transfer. I use PVDF membrane. Run at 70 V for ~ 3 hrs. After transfer my membrane becomes little dry and the outer coat of the membrane starts to come out leaving dark spots on the membrane. Also do protein ladder go bad? They have started to appear very dim in gel. Any suggestions!!
Thank you

-tech123-

If you post our protocol it will help - we literally have nothing to go on here!

-bob1-

This question might be a bit silly but did you soak the membrane in MeOH before using it? When I shifted from nitrocellulose to PVDF, I had no idea I was supposed to do that and ended up with having to repeat the transfer

-zodiac1505-

Hello:
I am trying to detect three proteins A, B and C. Unlike Immunohistochemistry, Why i cannot detect all the proteins in a single membrane when the antibodies against my protein are raised in different animals? I know the size of my proteins.
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